ORIGINAL RESEARCH
published: 19 May 2015
Edited by:
Flavia Vischi Winck,
Brazilian Center for Research in
Energy and Materials, Brazil
Reviewed by:
Bettina Scholz,
BioPol ehf./University of Akureyri,
Iceland
Klervi Le Lann,
Université de Bretagne Occidentale,
France
*Correspondence:
Peer M. Schenk,
Algae Biotechnology Laboratory,
School of Agriculture and Food
Sciences, University of Queensland,
Level 5, John Hines Building,
Brisbane, QLD 4072, Australia
p.schenk@uq.edu.au
Specialty section:
This article was submitted to
Plant Biotechnology,
a section of the journal
Frontiers in Plant Science
Received: 23 March 2015
Accepted: 05 May 2015
Published: 19 May 2015
Citation:
Duong VT, Thomas-Hall SR and
Schenk PM (2015) Growth and lipid
accumulation of microalgae from
fluctuating brackish and sea water
locations in South East
Queensland—Australia.
Front. Plant Sci. 6:359.
doi: 10.3389/fpls.2015.00359
Frontiers in Plant Science | www.frontiersin.org
doi: 10.3389/fpls.2015.00359
Growth and lipid accumulation of
microalgae from fluctuating brackish
and sea water locations in South
East Queensland—Australia
Van Thang Duong, Skye R. Thomas-Hall and Peer M. Schenk *
Algae Biotechnology Laboratory, School of Agriculture and Food Sciences, The University of Queensland, Brisbane, QLD,
Australia
One challenge constraining the use of microalgae in the food and biofuels industry is
growth and lipid accumulation. Microalgae with high growth characteristics are more
likely to originate from the local environment. However, to be commercially effective, in
addition to high growth microalgae must also have high lipid productivities and contain
the desired fatty acids for their intended use. We isolated microalgae from intertidal
locations in South East Queensland, Australia with adverse or fluctuating conditions,
as these may harbor more opportunistic strains with high lipid accumulation potential.
Screening was based on a standard protocol using growth rate and lipid accumulation as
well as prioritizing fatty acid profiles suitable for biodiesel or nutraceuticals. Using these
criteria, an initial selection of over 50 local microalgae strains from brackish and sea water
was reduced to 16 strains considered suitable for further investigation. Among these
16 strains, the ones most likely to be effective for biodiesel feedstock were Nitzschia
sp. CP3a, Tetraselmis sp. M8, Cymbella sp. CP2b, and Cylindrotheca closterium SI1c,
reaching growth rates of up to 0.53 day−1 and lipid productivities of 5.62 µg mL−1 day−1 .
Omega-3 fatty acids were found in some strains such as Nitzschia sp. CP2a, Nitzschia
sp. CP3a and Cylindrotheca closterium SI1c. These strains have potential for further
research as commercial food supplements.
Keywords: biodiesel, diatom, fatty acids, microalgae, omega-3 fatty acids
Introduction
Microalgae grow in most natural environments, typically aquatic and marine systems, but they
are also found in soil, ice, rock pools or in volcanic water that can have extreme environmental
fluctuations (Duong et al., 2012). Microalgae suffering from adverse or fluctuating conditions often
have the ability to accumulate higher contents of biochemical products for survival, such as lipid,
starch, protein or carotenoid contents (Lim et al., 2012). Microalgae can produce a variety of lipids
that can be used by the food and biofuel industry (Huerlimann et al., 2010). For the production
of biodiesel, fatty acids with a chain length of 14–18 carbons are preferable. Saturated fatty acids
C14, C16, and C18 and unsaturated fatty acids such as C16:1, C16:2, C18:1, and C18:2 are the most
important for producing good biodiesel quality (Schenk et al., 2008). This is because the other
unsaturated fatty acids with 3 or 4 double bonds have reduced stability in storage (Knothe, 2006;
Chisti, 2007).
1 May 2015 | Volume 6 | Article 359
Duong et al.
The components of fatty acids, including saturated and
unsaturated fatty acids produced by microalgae, differ for
different species and strains (Renaud et al., 1994; Salama et al.,
2013). The accumulation of fatty acid components can be
controlled by environmental conditions such as temperature,
nutrient availability and salinity (Renaud et al., 2002; Huerlimann
et al., 2010). Alternatively, the necessary fatty acids can be
developed using a chemical process. For example, unsaturated
fatty acids from microalgae, especially fatty acids with 3 or 4
double-bonds, can be easily hydrogenated under partial catalysts
(Jang et al., 2005), but it is preferable to identify and propagate
microalgae that do not require secondary treatment to modify
their fatty acid profile. Growth temperature is a major factor
that dramatically influences lipid accumulation in general or
lipid composition in particular. For instance, a higher growth
temperature can stimulate lipid accumulation in microalgae or
a lower growth temperature can lead to increased saturated fatty
acid composition (Renaud et al., 2002).
The screening of microalgae is not limited to those suitable
for biodiesel production, because microalgae also offer multiple
bio-products that can be used in a variety of industry (Schenk
et al., 2008; Huerlimann et al., 2010). For instance, unsaturated
fatty acids containing more than three double bonds, including
omega-3 fatty acids, are not preferable for biodiesel but they
can be useful as nutritious food or feed supplement. These
compounds currently have a high value and could compete with
fish oil in terms of productivity and environmental sustainability
(Adarme-Vega et al., 2014).
Although microalgae are known to produce considerable
amounts of lipids this it is not true for all microalgal strains.
The purpose of our research was to screen microalgal isolates
collected from brackish and sea water and identify those with
the highest growth and lipid production with desirable fatty acid
composition. As lipid accumulation capacity of algae changes
when subjected to adverse external factors, such as nutrient
supply and environmental conditions (Huerlimann et al., 2010;
Chen et al., 2011), the research focused on microalgae from sites
that have known adverse or fluctuating environmental conditions
(e.g., intertidal rock pools; Underwood, 1984). After isolating
pure algal cultures, growth and lipid production were compared
to establish which strains are most stable and productive and
most likely to be effective as a feedstock source for biofuel
production. In addition, strains which produce fatty acids
suitable for the health food industry were identified.
Materials and Methods
Microalgae samples were collected from a variety of sites in
the lower reaches of the Brisbane River, Stradbroke Island,
and Sunshine Coast in South East Queensland—Australia. The
samples represent different environmental conditions of tidal
brackish river water (50 cm depth), rock pools and beaches. Two
species from the Australian National Algae Culture Collection
from the Commonwealth Scientific and Industrial Research
Organisation (CSIRO) were used to compare growth and lipid
production with the strains that were isolated from the field
(Table 1). The microalgae were intentionally gathered from
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Characterization of Australian coastal microalgae
different environments to provide a diverse taxonomy, stored in
a cold box and transferred to the laboratory for analysis.
Isolation and Preliminary Screening
Microalgae were cultured in f/2 medium at 25 ± 1◦ C with a
12/12 h light/dark photoperiod at a light intensity of 120 µmol
photons m−2 s−1 from fluorescent lights (Osram L36W/840)
and constant bubbling conditions (LP-100 air pump, Shen-Zhen
Xingrisheng Industrial Co. Ltd) in a temperature-controlled
environment room. Single cells were isolated by a micropipette
on an inverted microscope and grown in 96 wells plates before
transferring to 100 mL flasks for pure cultivation as described
previously (Duong et al., 2012). The isolation procedure was
conducted with sterilized equipment and in the laminar flow.
Single cells were isolated by the micromanipulation method
and the pure culture was checked regularly for the presence
of contaminating algae or high abundance of bacteria during
inoculation and growth experiments. It should be mentioned that
the cultures in this study are not axenic. While it can be excluded
that they contain other microalgae or protists, they still contain
associated bacteria. This was considered desirable as microalgae-
associated bacteria often provide improved growth (Amin et al.,
2012). Algal strains were then provisionally screened based on
their ability for rapid growth and lipid fluorescence using Nile
red staining as described by Lim et al. (2012).
Classification by DNA Sequencing
Microalgal biomass was collected at the late exponential phase of
cultivation for DNA extraction. DNA extraction was performed
using a phenol:chloroform method. After extraction, genomic
DNA within the 18S rRNA region was amplified on a PCR
machine by using the following primers: Forward 5 -GCGGTA
ATTCCAGCTCCAATAGC-3 and Reverse 5 -GACCATACT
CCCCCCGGAACC-3 . The process followed was developed
by Lim et al. (2012). PCR templates were then purified by
using a Wizard SV Gel PCR Clean-Up System (Promega). For
sequencing preparation, 5 µL of a 25 ng µL−1 PCR product were
combined with 1 µL of a 10 µM solution of each of the above
primers. The reaction was topped up to 12 µL with Millipore
water in a 1.5 mL tube and sent to the Australian Genome
Research Facility (AGRF) at The University of Queensland for
sequencing and analysis. The DNA sequence data was compared
with Genbank entries for classification.
Standard Protocol for Growth Experiments
After obtaining pure cultures, all isolated strains were grown in
f/2 medium following a cultivation protocol that used bubbling
for aeration and mixing. The standard protocol can be briefly
described as follows: All strains were inoculated with 5 mL from
a recently grown master culture and cultured in 150 mL f/2
medium until the end of the exponential growth phase was
reached (less than 10% cell density increase/day) before starting
the standard growth experiment. This culture was then used
as the inoculum at a ratio of 1/10 for 300 mL f/2 medium in
400 mL bottles that were connected to a bubbling system and
exposed to 12/12 h light/dark photoperiod at a light intensity
of 120 µmol photons m−2 s−1 . Cells were counted daily by
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Duong et al. Characterization of Australian coastal microalgae

TABLE 1 | Sources and accessions of microalgae used in this study.

Species GPScoordinates Species origin and collection time Site status Genbank accession
number

Achnanthes sp. BR22.5_3
Bacillariophyta sp. B3
Bacillariophyta sp. SI1a
Chlorella sp. BR2
Cylindrotheca closterium SI1c
Cymbella cistuliformis CP2c
Cymbella sp. CP2b
Dunaliella tertiolecta
Navicula sp. BR22.52
Navicula sp. CP6a
Navicula sp. SI2d
Nitzschia sp. CP2a
Nitzschia sp. CP3a
Phaeodactylum tricornutum
Tetraselmis sp. M8
Thalassiosira rotula SI2a
27◦ 29 29.00S153◦ 00 48.00E Brisbane River (11.40 am 22/05/2012) Brackish water KF 360813
26◦ 48 12.11S153◦ 08 50.86E Bullcock Beach (3 pm 12/06/2011) Tidal rock pool KF 360815
27◦ 26 14.23S153◦ 30 51.08E Stradbroke Island (3 pm 07/07/2011) Brackish rook pool KF 360816
– Brisbane River Lim et al., 2012 Brackish water –
27◦ 26 14.23S153◦ 30 51.08E Stradbroke Island (3 pm 07/07/2011) Brackish rock pool KF 360818
24◦ 59 19.96S153◦ 21 04.74E Frazer Island, Champagne Pools (10 am 1/4/2012) Tidal rock pool KF 360819
24◦ 59 19.96S153◦ 21 04.74E Frazer Island, Champagne Pools (10 am 1/4/2012) Tidal rock pool KF 360820
– CSIRO Tasmania (CS-175/8) – –
27◦ 29 29.00S153◦ 00 48.00E Brisbane River (11.40 am 22/05/2012) Brackish water KF 360822
24◦ 59 19.96S153◦ 21 04.74E Frazer Island, Champagne Pools (10 am 1/4/2012) Tidal rock pool KF 360823
27◦ 25 33.82S153◦ 31 45.70E Stradbroke Island (3 pm 07/07/2011) Tidal rock pool KF 360824
24◦ 59 19.96S153◦ 21 04.74E Frazer Island, Champagne Pools (10 am 1/4/2012) Tidal rock pool KF 360825
24◦ 59 19.96S153◦ 21 04.74E Frazer Island, Champagne Pools (10 am 1/4/2012) Tidal rock pool KF 360826
– CSIRO Tasmania (CS-29/8) – –
– Rock Pool Maroochydore, Lim et al., 2012 Tidal rock pool JQ 423158
27◦ 25 33.82S153◦ 31 45.70E Stradbroke Island (3 pm 07/07/2011) Tidal rock pool KF 360828

using a hemocytometer. Nitrate concentrations were monitored
daily until the nutrient levels reached zero (below detection).
Nitrate was determined by using a colorimetric assay (API test
kit) and measured on a spectrophotometer at a wavelength
545 nm. Growth rates were calculated by the following equation
(Levasseur et al., 1993)
 ln N2
N1
K =
t2 − t1
where N1 and N2 equal cell counts at time 1 (t1 ) and time 2 (t2 ),
respectively. Doubling time was also calculated once the specific
growth rate was known.
ln2
Doubling time =
K
Microalgae were cultured for another 3 days after the nitrate
concentration in the medium became undetectable to induce
lipid biosynthesis.
Fatty Acid Methyl Esters (FAME) Analysis
Samples for FAME analysis were collected when lipid
accumulation reached its peak, after 3 days of starvation. A
total of 4 mL of microalgal culture was collected and centrifuged
at 8000 × g for 5 min. Biomass was collected and dried by a
vacuum pump for 30 min. Lipids in the microalgal pellet were
hydrolyzed and methyl-esterified in 300 µL of a 2% H2 SO4 and
methanol solution for 2 h at 80◦ C. Prior to the reaction, 50 µg of
heneicosanoic acid (Sigma, USA) was added as internal standard.
After the esterification step, 300 µL of 0.9% (w/v) NaCl solution
and 300 µL of hexane were added and mixed for 20 s. To separate
the phase, samples were centrifuged at 16,000 × g for 3 min. A
total of 1 µL of hexane layer was injected into an Agilent 6890 gas
chromatograph (GC) coupled to a 5975 MSD mass spectrometer
(MS). The running conditions were described by Agilent’s RTL
DBWax method (Brown, 1991).
Results
Growth of Selected Microalgal Strains
Out of 50 microalgal strains that were isolated, 16 were shortlisted
based on their rapid growth and high lipid fluorescence following
Nile red staining (Figure 1 shows an example). Strains were
then compared in standard growth and lipid accumulation
assays to determine the most suitable strains as feedstock
for biodiesel and/or nutraceuticals. The growth of microalgal
strains in the collection is presented in two groups for easier
comparison: diatoms and green microalgae. The fastest growing
strain Thalassiosira rotula SI2a grew 5.3 times faster than the
slowest growing strain Bacillariophyta sp. B3 (Table 2). Among
the diatomaceous species, Thalassiosira rotula SI2a’s cell density
was 2.47 × 106 cells mL−1 and this strain exhibited the highest
growth rate and lowest doubling time, achieving 0.64 day−1
and 1.09 days, respectively. The cell density of Phaeodactylum
tricornutum reached 6.97 × 106 cells mL−1 and grew more after
the standard assay’s completion. The growth rate and doubling
time of this strain was 0.49 day−1 and 1.42 days, respectively.
Cylindrotheca closterium SI1c’s growth reached a peak of 1.89 ×
106 cells mL−1 . Bacillariophyta sp. SI1a, Bacillariophyta sp. B3,
and Navicula sp. SI2d grew slowly and reached peaks of 3.18×105
cells mL−1 , 4.5 x 104 cells mL−1 and 1.18 × 105 cells mL−1 ,
respectively.
Dunaliella tertiolecta, Chlorella sp. BR2 and Tetraselmis sp.
M8 were the fastest growing green algal strains. Cell density of
three strains increased gradually and peaked after 7 to 9 days. The
cell density peaks for Chlorella sp. BR2, Tetraselmis sp. M8, and
Dunaliella tertiolecta were 2.46 × 106 cells mL−1 , 2.62 × 106 cells
mL−1 , and 2.67 × 106 cells mL−1 , respectively. These strains had
similar growth rates and doubling times (Table 2).

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Duong et al. Characterization of Australian coastal microalgae

TABLE 2 | Growth characteristics of the isolated strains in f/2 medium

during the duration of the standard assay.

Species Mean growth Doubling Maximum

rate (µ day−1 ) time (days) cell density
(x104 cells mL−1 )

Achnanthes sp. BR22.5_3 0.13 5.48 28.50

Bacillariophyta sp. B3 0.12 5.96 6.17
Bacillariophyta sp. SI1a 0.32 2.16 31.83
Chlorella sp. BR2 0.45 1.54 245.83
Cylindrotheca closterium SI1c 0.53 1.31 189.00
Cymbella cistuliformis CP2c 0.26 2.65 28.67
Cymbella sp. CP2b 0.53 1.31 103.17
Dunaliella tertiolecta 0.46 1.51 266.83
Navicula sp. BR22.52 0.34 2.05 24.50
Navicula sp. CP6a 0.39 1.80 42.33
Navicula sp. SI2d 0.29 2.40 11.83
Nitzschia sp. CP2a 0.22 3.08 298.33
20 µm Nitzschia sp. CP3a 0.17 3.99 123.83
Phaeodactylum tricornutum 0.49 1.42 696.67
FIGURE 1 | Cymbella sp. CP2b after Nile red staining under Tetraselmis sp. M8 0.43 1.60 262.17
fluorescence microscopy. Yellow droplets show lipid containing Thalassiosira rotula SI2a 0.64 1.09 247.00
triacylglycerides and orange droplets show autofluorescence from chlorophyll.
Cymbella sp. CP2b displayed fast growth rates and one of the highest lipid Shown are mean values from three separately-grown cultures each.
productivities (4.51 µg mL−1 day−1 ).
12.46% for Nitzschia sp. CP2a. DHA reached 1.11 and 1.36%
Nitrogen and phosphorous concentrations changed during for Nitzschia sp. CP3a and Nitzschia sp. CP2a, respectively. On
the cultivation of the strains. Nitrate concentration in the average, saturated fatty acids were the major FAMEs in most
medium decreased dramatically from day 3 to day 5 and nitrogen strains; the highest values were found in Thalassiosira rotula SI2a
depletion occurred from day 5 to day 7 of the experiment. (62.18%), Bacillariophyta sp. B3 (71.9%) and Navicula sp. CP6a
The concentration of phosphate decreased gradually over the (71.61%). However, total unsaturated fatty acids were higher than
experimental period. A depletion of phosphate occurred from total saturated fatty acids in strains such as Dunaliella tertiolecta
day 7 to day 10. Nitzschia sp. CP2a and Nitzschia sp. CP3a (62.08%), Cylindrotheca closterium SI1c (61.65%), and Nitzschia
consumed most nutrients among the strains tested (Figure 2). sp. CP3a (60.47%).

Lipid Contents in Microalgae Collection Discussion

The result from GC/MS showed that all selected microalgal
strains can produce fatty acids. Eighteen different fatty acids were
detected, however not all strains produced all 18 fatty acids. There
was no detection of some unsaturated fatty acid with 3 or 4
double bonds such as hexadecatrienoic acid, hexadecatetraenoic
acid, and stearidonic acid in Nitzschia sp. CP2a, Cymbella sp.
CP2b, Cymbella cistuliformis CP2c, Nitzschia sp. CP3a, and
Navicula sp. CP6a. Total FAMEs ranged from 9.27 to 50.53 µg
mL−1 . The highest FAME content was achieved for Nitzchia
sp. CP3a and the lowest was measured for Chlorella sp. BR2
(Figure 3). Palmitic acid and stearic acid were dominant among
the total fatty acids. The percentage of palmitic acid ranged from
19.83 to 37.65% in most of the strains, except for Bacillariophyta
sp. SI1a, Cylindrotheca closterium SI1c, Thalassiosira rotula SI2a,
and Navicula sp. SI2d. Stearic acid that accumulated in the strains
ranged from 14.81 to 57.73%, except for Nitzschia sp. CP2a,
Cymbella sp. CP2b, and Nitzschia sp. CP3a.
Interestingly, omega-3 eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) were found in most of the strains.
The highest EPA percentage of total FAMEs reached 30.85% for
Phaeodactylum tricornotum, 14.49% for Nitzschia sp. CP3a, and
All strains in the study were collected from estuary, coastal
waters and rock pools where environmental conditions change
frequently, especially changes of temperature, salinity, light
exposure, and nutrients. The changes require microalgae to
adapt and/or produce more biochemical products to ensure
their survival (Lim et al., 2012). Lipid is one of the biochemical
products that microalgae can produce along with starch under
nutrient deprived conditions in the light (Schenk et al., 2008).
Lipid productivities were highest for Nitzschia sp. CP3a (5.62 µg
mL−1 day−1 ), Tetraselmis sp. M8 (5.29 µg mL−1 day−1 ),
Cymbella sp. CP2b (4.51 µg mL−1 day−1 ), and Cylindrotheca
closterium SI1c (3.93 µg mL−1 day−1 ). The value for Tetraselmis
sp. M8 was higher than the lipid productivity previously reported
for this strain when grown under laboratory conditions (2.1 µg
mL−1 day−1 ; Lim et al., 2012). It is interesting to note that all of
these strains have been isolated from coastal rock pools subjected
to tides. Their high lipid productivities suggest that these
microalgae may be more opportunistic than others from more
stable environments (e.g., stationary lakes or open ocean). A
future study comparing the growth rates and lipid productivities

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Duong et al. Characterization of Australian coastal microalgae

A
500

400

Nitrate (mM)
300

200

100

0
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Nitzschia sp. CP2a Cymbella sp. CP2b Nitzschia sp. CP3a

B
70
60
50
Phosphate (mM)

40
30
20
10
0
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10

Nitzschia sp. CP2a Cymbella sp. CP2b Nitzschia sp. CP3a

FIGURE 2 | Nitrate (A) and phosphate (B) depletion in growth medium (daily uptake) of Cymbella sp. CP2b, Nitzschia sp. CP2a, and Nitzschia sp. CP3a.
Shown are mean values ± SEs from three separately-grown cultures each.

a
60
a
50
a a
-1

a
FAM E µ g m L

40
c c c bc c
b bd
30
bd d bd
20 b c
b b
10

0
B a c illa rio p h y ta s p . S I1 a

T h a la s s io s ira ro tu la S I2 a
T e tra s e lm is s p . M 8

D u n alie lla te rtio le c ta

B a c illa rio p h y ta s p . B 3

C h lo re lla s p . B R 2
N itzs c h ia s p . C P 2 a
N itzc h ia s p . C P 3 a

N a v ic u la s p . C P 6 a
N a v ic u la s p . S I2 d

C y lin d ro th e c a c lo s te riu m S I1 c
C y m b e lla c is tu lifo rm is C P 2 c
C y m b e lla s p . C P 2 b

P h a e o d a c ty lu m tric o rn o tu m

FIGURE 3 | Total fatty acid methyl esters (FAMEs) contents of Different letters above bars indicate statistically significant differences
the isolated microalgal strains, expressed in µg mL−1 . Shown (p < 0.05; Two-Way ANOVA, Tukey’s HSD test using GraphPad
are mean values ± SEs from three separately-grown cultures. Prism 6.0).

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 TABLE 3 | Fatty acid composition in percentage of total fatty acid methyl esters (FAMEs) of different microalgal strains collected in South East Queensland, Australia after cultivation to nitrate
depletion and an additional 3 days of starvation.
Duong et al.

Bacillariophyta
sp. B3
Bacillariophyta
sp. SI1a
Cylindrotheca
closterium
SI1c
Thalassiosira
rotula SI2a
Navicula
sp. SI2d
Tetraselmis
sp. M8
Phaeodactylum
tricornotum
Chlorella
sp. BR2
Dunaliella
tertiolecta
Nitzschia
sp. CP2a
Cymbella
sp. CP2b
Cymbella
cistuliformis
CP2c
Nitzschia
sp. CP3a
Navicula
sp. CP6a

Fatty acids

Lauric (C12:0) 1.12 1.64 1.59 2.17 2.18 0.95 0.13 2.05 0.27 0.00 0.00 0.00 0.00 0.00
Myristic (C14:0) 21.32 0.74 0.63 0.66 0.81 0.93 8.74 1.65 0.47 5.96 9.56 6.73 5.33 4.88
Palmitic (C16:0) 23.64 0.88 0.23 1.27 1.12 28.33 29.31 31.68 19.87 30.10 37.65 30.22 28.82 37.04

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Palmitoleic (C16:1) 0.45 1.43 4.66 1.19 1.37 1.79 0.21 4.71 0.35 31.57 38.26 21.31 27.28 15.75
Hexadecadienoic (C16:2) 5.08 13.72 8.97 3.18 6.56 14.59 0.43 1.42 1.44 2.80 1.60 2.49 3.33 1.63
Hexadecatrienoic (C16:3) 4.80 23.29 23.28 2.32 6.92 18.27 5.59 8.84 4.22 0.00 0.00 0.00 0.00 0.00
Hexadecatetraenoic (C16:4) 0.72 1.85 1.77 0.93 2.06 0.25 0.01 0.04 23.30 0.00 0.00 0.00 0.00 0.00
Stearic (C18:0) 25.15 39.13 35.21 57.73 52.32 19.79 14.81 29.28 16.65 5.71 6.56 20.39 5.25 28.74
Oleic (C18:1) 3.57 5.57 10.11 19.24 18.07 8.08 7.13 11.86 0.82 1.97 1.18 2.37 2.92 2.07
Linoleic (C18:2) 1.48 0.21 0.17 0.09 0.05 0.24 1.76 6.12 1.85 0.80 0.28 1.21 1.33 0.38
Linolenic (C18:3) 5.64 6.90 10.83 9.95 4.23 3.41 0.34 0.39 29.98 1.79 0.09 0.61 1.16 0.26
Stearidonic (C18:4) 0.10 0.15 0.20 0.10 0.15 0.14 0.00 0.13 0.00 0.00 0.00 0.00 0.00 0.00
Arachidic (C20:0) 0.52 0.18 0.42 0.17 0.11 0.26 0.60 1.28 0.57 0.19 0.20 0.77 0.13 0.95

6
Arachidonic (C20:4) 0.12 3.20 0.67 0.04 0.13 1.54 0.00 0.04 0.00 5.28 0.68 4.80 8.85 1.87
Eicosapentaenoic (EPA) (C20:5) 5.71 0.28 0.38 0.21 3.44 0.28 30.85 0.08 0.12 12.46 3.94 8.75 14.49 6.16
Behenic (C22:0) 0.15 0.40 0.27 0.18 0.19 0.34 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Docosatetraenoic (C22:4) 0.26 0.25 0.29 0.32 0.18 0.44 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Docosahexaenoic (DHA)(C22:6) 0.17 0.17 0.34 0.26 0.09 0.36 0.00 0.00 0.01 1.36 0.00 0.34 1.10 0.28
Saturated fatty acids (%) 71.90 42.96 38.35 62.18 56.73 50.60 53.59 65.93 37.82 41.96 53.97 58.11 39.53 71.61
Unsaturated fatty acids (%) 28.10 57.04 61.65 37.82 43.27 49.40 46.31 33.64 62.08 58.04 46.03 41.89 60.47 28.39
Total fatty acids (µg mL−1 ) 28.91 26.91 35.36 24.56 16.41 47.65 22.85 9.27 17.69 38.21 40.58 11.13 50.53 7.90
Lipid productivity (µg mL−1 day−1 ) 3.21 2.99 3.93 2.73 1.82 5.29 2.54 1.03 1.97 4.25 4.51 1.24 5.62 0.88

Shown are mean values from three separately-grown cultures each.

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Characterization of Australian coastal microalgae
Duong et al.
of microalgae isolated from stable and unstable environments
may further substantiate this notion. High growth rates of these
microalgae lead to rapid production of biomass while their high
lipid accumulation capability is likely to assist in higher survival
rates during adverse conditions. Interestingly, divergent from
this trend is Nitzschia sp. CP3a which despite its high lipid
productivity had a very slow growth rate. Further studies should
be conducted to test the survival rates of these strains and if these
correlate with their cellular lipid and/or starch contents.
Diatoms can survive in harsh environments (Seckbach, 2007)
and usually produce more lipids in harsher environments (Lim
et al., 2012). For this research, the difference of environmental
conditions between natural habitats and the laboratory may
have created a harsh environment that impacted on growth and
chemical accumulation of microalgae tested. Many of the strains
in this study adapted to their new environment and grew well
under laboratory conditions. For instance, Cymbella sp. is a
diatom that usually requires multi nutrients and minerals from
natural sources (Tarapchak et al., 1983; Takeda, 1998). However,
this strain grew well in f/2 medium and displayed one of the best
growth rates (Table 2). The indoor growth experiments were set
up for conditions similar to the natural habitats (temperature,
nutrients, light). However, the indoor conditions are not identical
to the natural conditions and the adaptability of the selected
microalgae to the laboratory condition is a useful indicator
of microalgae’s ability to adapt to different external conditions
which also will be encountered when grown at large scale for
commercial purposes.
In addition, nutrient availability also affects diatom growth.
For example, silica is the main components of the diatom
cell wall. Changing nutrient availability may directly influence
the structure of silica composition in the cell wall and affect
diatom growth (Tarapchak et al., 1983). Thus optimizing nutrient
availability can also substantially affect growth rates and biomass
production. For example, increasing of nutrients or nitrogen
leads to an increase of growth rate (Converti et al., 2009; Chen
et al., 2011; Borowitzka and Moheimani, 2013). However, to
enable a direct side-by-side comparison, a standard assay with
unoptimized parameters was used in the current study. Clearly,
higher growth rates and lipid productivities are achievable
after careful cultivation optimization. For example, this has
been achieved for Tetraselmis sp. M8 (Sharma et al., 2014). A
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Characterization of Australian coastal microalgae
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2015 Duong, Thomas-Hall and Schenk. This is an open-access article
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